Abstract

Background

Skipper butterflies (Hesperiidae) are a relatively well-studied family of Lepidoptera.
However, a combination of DNA barcodes, morphology, and natural history data has revealed
several cryptic species complexes within them. Here, we investigate three DNA barcode
lineages of what has been identified as Urbanus belli (Hesperiidae, Eudaminae) in Área de Conservación Guanacaste (ACG), northwestern Costa
Rica.

Results

Although no morphological traits appear to distinguish among the three, congruent
nuclear and mitochondrial lineage patterns show that “Urbanus belli” in ACG is a complex of three sympatric species. A single strain of Wolbachia present in two of the three cryptic species indicates that Urbanus segnestami Burns (formerly Urbanus belliDHJ01), Urbanus bernikerni Burns (formerly Urbanus belliDHJ02), and Urbanus ehakernae Burns (formerly Urbanus belliDHJ03) may be biologically separated by Wolbachia, as well as by their genetics. Use of parallel sequencing through 454-pyrosequencing
improved the utility of ITS2 as a phylogenetic marker and permitted examination of the intra- and interlineage
relationships of ITS2 variants within the species complex. Interlineage, intralineage and intragenomic
compensatory base pair changes were discovered in the secondary structure of ITS2.

Conclusion

These findings corroborate the existence of three cryptic species. Our confirmation
of a novel cryptic species complex, initially suggested by DNA barcode lineages, argues
for using a multi-marker approach coupled with next-generation sequencing for exploration
of other suspected species complexes.